CN104562213A - Amplification sublibrary and construction method thereof - Google Patents
Amplification sublibrary and construction method thereof Download PDFInfo
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Abstract
The invention provides an amplification sublibrary and a construction method thereof. The construction method comprises the following steps: S1, performing PCR amplification on target fragments of multiple samples to obtain a plurality of amplification products; S2, performing balanced mixing on the amplification products to obtain a mixed product; S3, sequentially performing fragmentation and terminal repairing on the mixed product, and adding A at a terminal 3' of the mixed product to obtain an A-repaired product; S4, jointing the A-repaired product by using a joint of PCR-free to obtain the amplification sublibrary. Through balanced mixing of the amplification products and through joint connection by using the optimized PCR-free, by the construction method, preference generated by PCR as well as chimeras in environmental samples is avoided, so that sequencing data between different samples have good consistency, subsequent clustering analysis and technical repeatability are more reasonable and more accurate, and can more really reflect evolutional relationship between the environmental samples.
Description
Technical field
The present invention relates to high-flux sequence field, in particular to a kind of amplicon library and construction process thereof.
Background technology
The quick acquisition of life entity genetic information has very important meaning for the research of life science.First-generation sequenator, to the dependence of electrophoretic separation technique, makes it be difficult to improve further speed and the parallelization degree of analysis, is also difficult to reduce order-checking cost by microminiaturization.Through continuous technological development and improvement, at the beginning of 21 century, be born with the s-generation sequencing technologies of the Solid technology of the Genome Analyzer technology of Roche 454 technology, illumina company and ABI company for mark.Second-generation technology greatly reduces order-checking cost, has also increased substantially order-checking speed and has maintained high accuracy.Wherein, the First sequenator of illumina company came out in 2006, develop a series of sequenator afterwards, as Hiseq2000, Hiseq2500, Miseq, Hiseq X etc., in accuracy, flux, cost and speed, performance is more outstanding, and becomes the maximum s-generation sequenator of global usage quantity.While s-generation order-checking platform is constantly perfect, to carry out third generation sequencing technologies that non-PCR order-checking is principal character also first aobvious clue to unique DNA, but wait to solve due to its key technical problem, be not also used widely at present.
In environment, the structure of community of microorganism and diversity are the study hotspots of microbial ecology.The research of microbial diversity relates to the numerous areas such as agricultural, soil, forestry, ocean, mine, human medical.The experimental implementation that has tradition research means wastes time and energy, not Culturability, trace microorganism cannot be detected, unknown etc. limitation cannot be explored.The maturation of s-generation high throughput sequencing technologies (especially illumina Miseq high throughput sequencing technologies) is with universal, enable us to carry out degree of depth order-checking to environmental microorganism, the difference of testing environment sample in bacterium, fungi, ancient bacterium classification between species, accurately instructs the formation of varying environment microorganism delicately.
Amplicon order-checking is checked order to the PCR primer of length-specific or the fragment of catching, and not only comprises 16S rDNA and check order, also comprises that 18S rDNA checks order, ITS checks order and functional gene detection etc.Illumina MiSeq s-generation high-flux sequence platform is adopted to carry out certain hypervariable region degree of depth order-checking of 16S/18S/ITS to deriving from varying environment sample, the extremely faint change that environmental microorganism structure of community occurs with the change of external environment can be detected delicately, for the relation of our microorganisms and environment, the utilization of environmental improvement and Microbial resources has important theory and realistic meaning.
16S rDNA is the DNA sequence dna of encoding bacterial small subunit ribosome, and molecular size is about 1540bp, is made up of 9 variable regions and 10 conserved regions cross arrangements.Conserved regions energy reflection Interspecific relationship, variable region there are differences between different strain.According to conserved regions primers, is increased out in variable region and check order, by the comparison of sequencing data and associated databases, the position of microorganism in evolutionary tree can be determined, thus the microbe species that may exist in qualification sample.Research shows, it is comparatively accurate that V4 target genetic region (about 300bp) classifies to microorganism.
Banking process based on the 16S V4 district amplicon sample of illumina order-checking platform mainly contains three kinds at present.First method, for use comparatively early and pcr amplification builds storehouse method widely, namely comprises pcr amplification step building in the process of storehouse.First mix products is cut glue purification; Then carry out end reparation successively, add " A ", add Pair-end joint (introducing illumina sequencing primer sequence at sequence two ends) and purifying; Last is that template carries out pcr amplification reaction (introducing complete illumina joint sequence, index sequence and sequencing primer sequence) with biased sample, again after purifying, can complete library construction.
Second method is the step TRAP that illumina official is recommended, and is namely completed by One_step PCR and builds storehouse process.The method uses and comprises 16S V4 district upstream conserved sequence and illumina P5 end connector sequence as upstream primer, 16S V4 district downstream conserved sequence, index sequence and illumina P7 end connector sequence carry out pcr amplification as downstream primer, can complete and build storehouse after product mixes pond.
The third method is that Truseq PCR-free builds storehouse method, namely applies Truseq PCR-free test kit completely and builds library.First mix products is cut glue purification; Then carry out end reparation successively, add " A ", add PCR-free joint (connecting complete illumina joint sequence, index sequence and sequencing primer sequence); After last magnetic beads for purifying, library construction can be completed.
In above-mentioned construction process, purifying is carried out to the pcr amplification product carrying sequence label and quantitative method mainly contains two kinds.First method is after pcr amplification, adopts PCR primer to reclaim kits, utilizes spectrophotometer quantitative, then equimolar amount mixing; Second method is after pcr amplification, PCR primer cut separately glue reclaim and Qubit quantitative, then equimolar amount mixes.
Wherein, the quantitative homogenization method of the first purifying cannot remove the primer dimer in PCR primer, affect the accurate quantitative analysis of spectrophotometer to target fragment, make cannot mix by equimolar amount veritably between sample, and make the homogeneity of sequencing data poor, add survey sample proportion higher, the project cycle cannot ensure; The homogenization method that the second purifying is quantitative, glue organic efficiency is lower, causes PCR primer to be lost more, makes to build storehouse initial amount and cannot ensure in normal range, and then Kucheng's power is built in impact.Complex operation simultaneously, reagent cost is higher.
But, when adopting above-mentioned pcr amplification banking process to build libraries such as such as 16S rDNA or ITS, because 16S rDNA or ITS etc. uses hybrid template in amplification procedure, and the similarity of sequence is very high between these templates, so genome DNA easily occurs mistake when 16S rDNA increases, produce some in the environment originally and non-existent " 16S rDNA " sequence.Wherein the structure impact of chimeria (mosaic) and heteroduplex (heteroduplex) on library is larger.Mosaic in the process of PCR, from the 16S rDNA of different microorganisms, coamplification occurs cause, when increasing for template with the 16S rDNA of microorganism A, likely owing to extending not exclusively, produce the incomplete 16SrDNA single chain molecule of microorganism A, and when next round pcr amplification, it just may be annealed with the 16S rDNA molecule of the microorganism B of another kind of Sequences similar, and self as primer with the 16S rDNA molecule of microorganism B for template extends, produce a part and come from microorganism A and another part comes from the mosaic 16S rDNA molecule of microorganism B, this is just called mosaic.In this primary sample and non-existent chimer molecules, the diversity of microorganism in our too high estimation environment can be made.Heteroduplex is after PCR reaction enters plateau, because the ratio between primer and template sharply reduces, the probability of annealing between primer and template is caused to reduce, and the probability of annealing between the template of Sequences similar increases, anneal between the 16S rDNA molecule of these Sequences similar and just define heteroduplex.Information analysis shows, this banking process makes the biology cluster between sample unreasonable (having the phenomenon that library is convergent), the technology poor repeatability of same sample.
And employing one step amplification build storehouse method storehouse is built to above-mentioned sample time, when upper machine order-checking, the reagent that illumina Miseq can not be used to match checks order, need self-defined sequencing primer, namely synthesis 16S V4 district upstream conserved sequence holds sequencing primer as P5 separately, downstream, 16S V4 district conserved sequence primer holds sequencing primer as P7, and the reverse sequence of 16S V4 district downstream conserved sequence, as the sequencing primer of index, needs to synthesize special Phix library simultaneously.On one step amplification library during machine order-checking, can only mix with the amplicon library of identical variable region is operation passage (run), so not only affects row's machine order-checking handiness, and cannot ensure sequencing quality.
Truseq PCR-free builds the reagent that storehouse method can directly use Illumina Miseq to match and checks order, and biology cluster is reasonable between sample, same sample reproducible, but reagent cost higher (TruSeq DNA PCR-Free SamplePreparation Kit), needs to capture its technology barriers.
Therefore, how to provide a kind of simple to operate, cost is low and the amplicon library constructing method that sequencing data homogeneity between the different samples obtained is good has become a technical problem urgently to be resolved hurrily.
Summary of the invention
Main purpose of the present invention is to provide a kind of amplicon library and construction process thereof, to improve the homogeneity of constructed library gained sequencing data after order-checking.
To achieve these goals, according to an aspect of the present invention, provide a kind of construction process of amplicon library, this construction process comprises the following steps: S1, carries out pcr amplification respectively, obtain multiple amplified production to the object fragment of multiple sample; S2, carries out balanced mix to multiple amplified production, obtains mix products; S3, carries out end reparation successively to mix products and 3 ' end adds " A ", obtains band " A " and repairs product; S4, adopts the joint of PCR-free to repair product to band " A " and carries out joint connection, obtain amplicon library.
Further, in step S1, adopt the object fragment of amplimer to multiple sample with the special sequence label of sample to carry out pcr amplification, obtain amplified production.
Further, after step S1, and before step S2, also comprise the step of multiple amplified production being carried out to purifying, preferably adopt magnetic bead to carry out purifying to multiple amplified production; More preferably isopyknic magnetic bead is adopted to carry out purifying to multiple amplified production.
Further, after purification, also comprise and quantitative step is carried out to multiple amplified production, preferably adopt spectrophotometer to carry out quantitatively multiple amplified production.
Further, upon step s 2, and before step S3, also comprise the step that electrophoresis cuts glue purification is carried out to mix products.
Further, in step s3, employing The NEBNext Ultra End Repair/dA-Tailing Module carries out end reparation and 3 ' end adds " A ", obtains band " A " and repairs product.
Further, the joint of PCR-free comprises Illumina joint sequence, library sequence label and object sequencing fragment primer sequence.
Further, in step s 4 which, adopt the joint of the PCR-free in Truseq test kit to carry out joint connection, obtain amplicon library.
Further, after joint Connection Step, also comprise the step of belt lacing fragment being carried out to purifying.
According to a further aspect in the invention, provide a kind of amplicon library, adopt any one construction process above-mentioned to build and form.
Apply technical scheme of the present invention, by amplified production being carried out balanced mix and using the PCR-free optimized to carry out joint connection, make amplicon library constructing method of the present invention not only in low cost, the homogeneity of sequencing data is achieved under the prerequisite of low Operating Complexity, and avoid PCR produce Preference and environmental samples in mosaic, make the sequencing data homogeneity between different sample good, and then make follow-up cluster analysis and technology repeatability more reasonable, more accurate, it is a kind of method being applied to amplicon homogeneity library construction of optimization, constructed library more can the evolutionary relationship of reaction environment sample room truly.
Accompanying drawing explanation
The Figure of description forming a application's part is used to provide a further understanding of the present invention, and schematic description and description of the present invention, for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 shows the schematic flow sheet according to amplicon library construction in a kind of typical embodiment of the present invention;
Fig. 2 shows the schematic flow sheet according to amplicon library construction in the another kind of preferred embodiment of the present invention;
Fig. 3 shows the electrophoresis detection figure according to the amplified production in a kind of typical embodiment of the present invention after pcr amplification;
Fig. 4 shows the result figure according to carrying out storehouse inspection in a kind of typical embodiment of the present invention to constructed amplicon library;
Fig. 5 shows the Clustering Effect figure of the amplicon library the data obtained adopted constructed by a kind of method of the prior art;
Fig. 6 shows the Clustering Effect figure of the amplicon library the data obtained adopted constructed by another kind of method of the prior art; And
Fig. 7 shows the Clustering Effect figure of the amplicon library the data obtained adopted constructed by method of the present invention.
Embodiment
It should be noted that, when not conflicting, the embodiment in the application and the feature in embodiment can combine mutually.Below with reference to the accompanying drawings and describe the present invention in detail in conjunction with the embodiments.
Mention as background technology part, amplicon library constructing method of the prior art, when the amplicon library of especially carrying out certain hypervariable region of 16S/18S/ITS to the sample deriving from varying environment builds, there is the problem of the sequencing data homogeneity difference of the high and each sample of complicated operation, cost.Solving the problem to improve, in a kind of typical enforcement side of the present invention, providing a kind of construction process of amplicon library, as shown in Figure 1, this construction process comprises the following steps: S1, carries out pcr amplification, obtain multiple amplified production to the object fragment of multiple sample; S2, carries out balanced mix to multiple amplified production, obtains mix products; S3, carries out fragmentation, end reparation and 3 ' end successively to mix products and adds " A ", obtains band " A " and repairs product; S4, adopts the joint of PCR-free to repair product to band " A " and carries out joint connection, obtain amplicon library.
Above-mentioned construction process of the present invention, by amplified production being carried out balanced mix and using the PCR-free optimized to carry out joint connection, make amplicon library constructing method of the present invention not only in low cost, the homogeneity of sequencing data is achieved under the prerequisite of low Operating Complexity, and avoid PCR produce Preference and environmental samples in mosaic, make the sequencing data homogeneity between different sample good, and then make follow-up cluster analysis and technology repeatability more reasonable, more accurate, it is a kind of method being applied to amplicon homogeneity library construction of optimization, constructed library more can the evolutionary relationship of reaction environment sample room truly.
In above-mentioned construction process of the present invention, the sample deriving from varying environment being checked order, in order to distinguish the environmental sources of each sample, usually needing to mark the sample deriving from varying environment.In the present invention, preferably in above-mentioned steps S1, adopt the object fragment of amplimer to multiple sample with the special sequence label of sample to carry out pcr amplification, obtain multiple amplified production.In actual library construction, be increase the base sequence of one section of 6-12bp as sample label sequence in the outside of object fragment amplification primer, in different sample, this sample label sequence is different.Be convenient to multiple sample to mix like this carry out follow-up library construction step, when extensive sample carries out amplicon library construction, can be time saving and energy saving, increase work efficiency.
In above-mentioned construction process of the present invention, the method that amplified production carries out balanced mix has multiple, such as can the amount of amplified production of each sample of electrophoresis detection, then carry out balanced mix according to the brightness power of the amplified band of each amplified production or adopt spectrophotometer to carry out measurement of concetration to each amplified production, then carrying out balanced mix.In order to mix more accurately, to improve the homogeneity of each sample sequencing data.In the present invention, after step S1, and before step S2, also comprise the step of multiple amplified production being carried out to purifying, preferably adopt magnetic bead to carry out purifying to multiple amplified production.In a kind of preferred embodiment of the present invention, as shown in Figure 2, isopyknic magnetic bead is adopted to carry out purifying to multiple amplified production.
In the present invention, employing magnetic beads for purifying can high efficiente callback object fragment.And adopting isopyknic magnetic bead to carry out purifying to multiple amplified production can also carry out high efficiente callback for selecting the object fragment of specific size.Adopt isopyknic magnetic beads for purifying to have no to use in the homogenization purification step in biological amplicon library construction field, the present invention carries out purifying by adopting isopyknic magnetic bead to multiple amplified production, the interference of primer dimer can not only be removed, and the impact of some impurity such as RNA, albumen can also be removed simultaneously, what follow-up balanced mix step can be mixed is more accurate.In the present invention, after purification step, also comprise and quantitative step is carried out to multiple amplified production, preferably adopt spectrophotometer to carry out quantitatively multiple amplified production.Conventional spectrophotometer, such as NanoDrop or Qubit can measure the concentration of amplified production fast, simply, exactly, to realize the equimolar amount mixing between different sample.
In above-mentioned construction process of the present invention, after the amplified production of the sample deriving from multiple environment is mixed, just can carry out follow-up end reparation and add step A.In order to improve remediation efficiency further, improving and building storehouse quality, in a kind of preferred embodiment of the present invention, upon step s 2, and before step S3, also comprise and the step that electrophoresis cuts glue purification is carried out to mix products.The mode adopting electrophoresis to cut glue purification carries out purifying to mix products, suitable size amplified production can be selected to carry out subsequent operations on the one hand, the non-targeted fragments such as primer dimer, proteolytic enzyme and the non-specific fragment in amplification step can also be removed on the other hand, make object fragment purer.
In above-mentioned construction process of the present invention, object fragment is pcr amplification product, although compare the DNA fragmentation that the ultrasonic mode interrupted obtains there is less protruding terminus or Single-stranded DNA fragments, but in order to prevent the incomplete double-strand that may exist in PCR process, thus also need the step of the amplified production of above-mentioned mixing being carried out to end reparation.Add in " A " step in above-mentioned end reparation and 3 ' end, conventional step can be adopted to carry out, as long as above-mentioned mixing fragment can be made to carry out end reparation and add " A " at its 3 ' end.In the present invention, preferred employing The NEBNext Ultra End Repair/dA-Tailing Module carries out end reparation and 3 ' end adds " A ", obtains band " A " and repairs product.Mentioned reagent in the library construction Kit of NEB company can make end reparation and 3 ' end add " A " one step complete, and repair and the joint efficiency of " A " higher, it is higher that production concentration repaired by the band " A " obtained.
In above-mentioned construction process of the present invention, in step s 4 which, adopt the joint of the PCR-free in Truseq test kit to carry out joint connection, obtain amplicon library.Adopt the joint of the PCR-free in Truseq test kit, the library that builds can not only be obtained by single stepping, and owing to adopting the method for non-PCR amplification to obtain library, the object fragment avoided in varying environment sample forms mosaic when pcr amplification, thus the artificial polymorphism introducing microorganism.In mentioned reagent box, the joint of PCR-free comprises Illumina joint sequence, library sequence label and object sequencing fragment primer sequence.Use the PCR-free joint with above-mentioned three kinds of sequences composition, the reagent that can match with Illumina Miseq checks order, and in the sequencing data obtained between sample biology cluster reasonable, same sample reproducible.
In a kind of preferred embodiment of the present invention, employ NEB and build storehouse test kit and Truseq PCR-free joint, this had not all reported in existing patent documentation with the article delivered.In the present invention, library constructed by the banking process that both discoveries combine not only has the feature (qualification rate more than 98% that stability is high and qualification rate is high, maintain an equal level with the qualification rate of Truseq PCR-free test kit), and require low to sample initial amount, it is short to build the storehouse operating time, and technical complexity is low.
In above-mentioned construction process of the present invention, the amplicon library comprising object fragment can be obtained after joint Connection Step.In order to improve the purity in library further, in a kind of preferred embodiment of the present invention, as shown in Figure 2, the step of belt lacing fragment being carried out to purifying is also comprised after joint connects, 0.8 × Ampure XP magnetic bead can be adopted to carry out purifying, again can select the size in library after band top connection, obtain the amplicon library of object size enrichment more.
In the another kind of typical embodiment of the present invention, provide a kind of amplicon library, this amplicon library adopts any one library constructing method above-mentioned to build and forms.Because storehouse process of building of the present invention does not have pcr amplification step, can not introduce mosaic and heterodimer, make the technology of same sample reproducible, the sequencing data homogeneity between different sample is good, and the biology cluster between sample is reasonable.
Further illustrate beneficial effect of the present invention below in conjunction with specific embodiments.
(1) soil in rare-earth mineral soil, coal gangue soil, oil tea soil, oil tea fertilized soil, photosynthetic bacteria liquid and Japanese microbial inoculum environment is collected, to classify to the microbiological specimens in soil or to carry out evolutionary analysis.Adopt soil genome DNA extracting reagent kit (DP336, sky root), from above-mentioned pedotheque, extract the DNA of microorganism.
(2) agarose gel electrophoresis is used, in conjunction with the integrity of Qubit detection by quantitative genomic dna, concentration and pollution condition.If there is a large amount of RNA in sample to need to use RNase digestion.
(3) DNA concentration per sample, dilutes sample, uses sterilized water dilute sample to 1ng/ μ l.
(4) the hypervariable region V4 of 16S rDNA is increased.Upstream and downstream amplimer is made up of general object fragment amplification primer and sample label sequence.Wherein, general forward primer sequence is SEQ ID NO.1:F-GTGCCAGCMGCCGCGGTAA; Reverse primer is SEQ ID NO.2:R-GGACTACHVGGGTWTCTAAT; Each sample carries special sequence label and specifically sees the following form 1.Amplification reaction system following (30 μ l):
Table 1:
(5) response procedures is as follows:
(6) each sample amplification two technology repeat, and are mixed into a pipe before detection.
(7) electrophoresis detection PCR primer.Get 3 μ l to each sample and carry out product detection, 16S V4 district is about 300bp, detects as Fig. 3.In Fig. 3, intermediate strap is the large tick marks of DNA molecular, and 5 molecule markers are from the bottom to top followed successively by 100bp, 200bp, 300bp, 400bp and 500bp, can find out, the size of PCR primer is consistent with the object clip size of expection.
(8) equal-volume magnetic beads for purifying pcr amplification product.20 μ l amplified productions got by each sample, add 20 μ l magnetic beads respectively and carry out purifying (as Agencourt AMPure XP magnetic bead, Beckman), then dissolve with sterilized water.
(9) the quantitatively rear equimolar amount mixing of spectrophotometer.Spectrophotometer quantitatively (as Nanodrop) is carried out respectively to the amplified production after each purifying, and the amplified production of same object fragment is got identical mass mixing obtains mix products.Owing to can be that the amplified production of same clip size is mixed into a library, so finally can realize equimolar amount during mixing.
(10) the object fragment in purifying mix products also completes follow-up library construction step by the PCR-free method that the present invention optimizes.
After mix products is carried out electrophoresis, the glue carrying out object fragment with QIAquick Gel Extraction Kit (Qiagen) reclaims;
Then the The NEBNext Ultra End Repair/dA-Tailing Module of NEB company and The NEBNextUltra Ligation Module reagent is adopted to carry out end reparation and add " A ";
The joint (comprising complete illumina joint sequence, index sequence and sequencing primer sequence) of the PCR-free in Truseq test kit is adopted to carry out the connection of PCR-free joint;
After adding joint, with 0.8 × Ampure XP magnetic bead, purifying is carried out to sample.
(11) carry out storehouse inspection to above-mentioned library, detected result is shown in Fig. 4.Because PCR-free library institute connecting joint is connection wye, the more common flat end library of mobility is low, and from the detection figure of the amplified production shown in Fig. 3, the Insert Fragment in above-mentioned library is about 300bp, therefore according to the storehouse inspection experience in PCR-free library, in Fig. 4,574 peaks being depicted as the fragment connecting single-ended joint, 1156 peaks being depicted as the fragment connecting both-end joint.In this storehouse inspection figure, only there are two main peaks, show that the library constructed by the present invention does not have joint to pollute, there is the peak shape feature in the library of PCR-free.All the other libraries also have identical peak shape feature, thus insert sheet degree qualified.Miseq can be used after qualified to carry out upper machine order-checking.
(12) information analysis is carried out to the data obtained, and compare with the sequencing data in the amplicon library constructed by the banking process of prior art, the comparative result of (raw tag) sequence number (combined tags) of the spliced tape label of different sample institute's output in the library constructed by different methods is in table 2.
Table 2:
Table 2 is the raw data of sequence number after the splicing of all samples in embodiment.As can be seen from Table 2, not carry out quantitatively or compared with banking process that purifying is not quantitative without purifying with prior art, adopt the library constructed by banking process of the present invention, in the sequence number of obtained spliced tape label (tag), respectively have quality.Whether the data weighing each sample in sequencing data have homogeneity, also need to carry out statistical analysis to the index of the homogeneity in the library constructed by different methods.Data in table 2 are obtained 3 kinds of homogeneity indexs of quantivative approach by statistical study, comprise the variation coefficient, be less than the sample proportion of 10,000 sequences (reads), sample proportion in upper and lower 30% scope of mean value, statistics is as shown in table 3.
Table 3:
By three homogeneity indexs of different basis weights method in contrast table 3, can find out: the variation coefficient (being only about 7%) of " magnetic beads for purifying Qubit is quantitative " and " magnetic beads for purifying Nanodrop is quantitative " method is significantly lower than " not purifying non-quantitative " and " purifying Qubit is not quantitative " (up to more than 44%) method; And the sample proportion be less than in the sample proportion of 1W bar reads and upper and lower 30% scope of mean value is significantly higher than the latter.These three homogeneity indexs do not have significant difference between " magnetic beads for purifying Qubit is quantitative " and " magnetic beads for purifying Nanodrop is quantitative " two kinds of methods.Visible, adopt the library constructed by method of the present invention to achieve unforeseeable technique effect in the data homogeneity of output.And the reagent cost of " magnetic beads for purifying Nanodrop is quantitative " method is lower, it is the method being best suited for the extensive library construction of high-quality in these 3 kinds of quantivative approachs.
Further, contriver also compares the characteristic of sequencing data in cluster analysis in the library constructed by method of the present invention and the library constructed by prior art.
First, selected the soil of above-mentioned table 2 middle-weight rare earths ore deposit, bastard coal stone ore and oil tea soil three kinds of different sourcess, carried out cluster analysis to the sequencing data of species in the soil of three kinds of different sourcess, concrete cluster analysis result is as shown in Fig. 5,6 and 7.In figures 5,6 and 7, A, B, C represent the numbering of the soil of above-mentioned 3 three kinds of different sourcess respectively, the little group # (divide into groups according to the different geographical of often kind of soil, the species composition of the different group of soil of the same race has notable difference) that letter the 1st numeral is below divided into groups to often kind of soil; Sample number into spectrum in 2nd each group of numeral, the 3rd numeral be technology repeat to number (namely above two numerals the same be same sample), the 4th numeral is bank number.
The cluster analysis result of the sequencing data in library constructed by a step amplification banking process is adopted to see Fig. 4.In the diagram, from the cluster analysis of each group deriving from same soil: B-5-1-1-2 and the B-5-2-1-2 species composition " B-5 " group is close, and thus cluster together; Equally, in " C-2 " group, together with the close and cluster of the species composition of C-2-1-1-1, C-2-3-1-2, C-2-2-1-1 and C-2-2-2-2 tri-samples.Cluster from the technology of sample repeats: " A-2-2-1-1 " and " A-2-2-2-2 ", " B-1-2-1-1 " are with " B-1-2-2-2 ", " C-2-2-1-1 " and " C-2-2-2-2 " these 3 samples, between each technology repeats, because species composition is close, can cluster between two.This cluster analysis result shows: the banking process of a step amplification can realize more rational cluster, and technology is reproducible.
The cluster analysis result adopting pcr amplification to build the sequencing data in library constructed by the method for storehouse is shown in Fig. 6.In figure 6, the cluster analysis of each group from deriving from same soil: the sample of " A-2 " group due to point two libraries respectively and " A-1 ", " A-3 " group get together according to library, " C-2 " group also shows the similar feature according to library cluster.Cluster from sample technology repeats: only have " B-1-2-6-7 " to repeat to get together with the technology of " B-1-2-7-8 " this 1 sample, " A-2-2-6-7 " and " A-2-2-7-8 ", " C-2-2-6-7 " and " C-2-2-7-8 " all can not clusters between two, and show the feature according to library cluster.This cluster analysis result shows: what pcr amplification banking process embodied is according to library cluster, but not carries out cluster according to sample is similar, and thus cluster is unreasonable, technology poor repeatability, has the phenomenon that library is convergent.
The cluster analysis result adopting PCR-free to build the sequencing data in library constructed by the method for storehouse is shown in Fig. 7.In the figure 7, from the cluster analysis of each group deriving from same soil: " A-2 " group, " B-2 " group and " C-2 " group, because species composition is close, have 5,3 and 4 samples to get together respectively.Cluster from sample technology repeats: " A-2-2-8-9 " and " A-2-2-9-10 ", " B-1-2-8-9 " are with " B-1-2-9-10 ", " C-2-2-8-9 " and " C-2-2-9-10 " these 3 samples, because species composition is close between each technology repeats, can cluster between two.This cluster analysis result shows: the cluster that PCR-free builds storehouse method is reasonable, and technology is reproducible, more meets biology grouping than the cluster of a step amplification banking process.
Preliminary conclusion as can be seen from above-mentioned 3 kinds of banking process cluster analyses: pcr amplification builds the cluster result of storehouse method and Biological background is misfitted; The increase cluster result of building storehouse method of the banking process of PCR-free of the present invention and 1 step of prior art meets biology and expects, but the technology of the banking process of PCR-free of the present invention repeatability is better.
As can be seen from the above description, the above embodiments of the present invention achieve following technique effect:
(1) the homogenization method adopting magnetic beads for purifying quantitatively to combine with spectrophotometer: reagent cost is lower, simple to operate, organic efficiency is high, equal-volume magnetic beads for purifying can remove the primer dimer in PCR primer, make quantitatively more accurate to object fragment of spectrophotometer, improve the homogeneity of sequencing data.
(2) the PCR-free joint optimized is adopted to connect: simple to operate, save time, reagent cost is lower, and the reagent that can directly use illumina Miseq to match checks order.There is no pcr amplification step owing to building storehouse process, mosaic and heterodimer can not be introduced.And sequencing data shows by analysis, the technology of same sample is reproducible, and the biology cluster between sample is reasonable.
Visible, above-mentioned construction process of the present invention is combined by operation is comparatively simple, that organic efficiency is high purification process and accuracy is good, cost is low quantivative approach, improves the homogeneity of sequencing data between multiple sample; Adopt the PCR-free joint of optimization to connect simultaneously, not only increase the technology repeatability of the biology cluster reasonableness between sample, same sample, and reduce reagent cost, make the order-checking of amplicon library construction, upper machine and information analysis more quick and accurate.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. the construction process in amplicon library, is characterized in that, described construction process comprises the following steps:
S1, carries out pcr amplification respectively to the object fragment of multiple sample, obtains multiple amplified production;
S2, carries out balanced mix to described multiple amplified production, obtains mix products;
S3, carries out end reparation successively to described mix products and 3 ' end adds " A ", obtains band " A " and repairs product;
S4, adopts the joint of PCR-free to repair product to described band " A " and carries out joint connection, obtain described amplicon library.
2. construction process according to claim 1, is characterized in that, in described step S1, adopts the object fragment of amplimer to described multiple sample with the special sequence label of sample to carry out pcr amplification respectively, obtains described multiple amplified production.
3. construction process according to claim 1, is characterized in that, after described step S1, and before described step S2, also comprises the step of described multiple amplified production being carried out to purifying, preferably adopts magnetic bead to carry out purifying to described multiple amplified production; More preferably isopyknic magnetic bead is adopted to carry out purifying to described multiple amplified production.
4. construction process according to claim 3, is characterized in that, after the step of described purifying, also comprises and carries out quantitative step to described multiple amplified production, preferably adopts spectrophotometer to carry out quantitatively described multiple amplified production.
5. construction process according to claim 1, is characterized in that, after described step S2, and before described step S3, also comprises and carries out to described mix products the step that electrophoresis cuts glue purification.
6. construction process according to claim 1, is characterized in that, in described step S3, employing The NEBNext UltraEnd Repair/dA-Tailing Module carries out end reparation and 3 ' end adds " A ", obtains described band " A " and repairs product.
7. construction process according to claim 1, is characterized in that, the joint of described PCR-free comprises Illumina joint sequence, library sequence label and object sequencing fragment primer sequence.
8. construction process according to claim 1, is characterized in that, in described step S4, adopts the joint of the PCR-free in Truseq test kit to carry out joint connection, obtains described amplicon library.
9. the construction process according to claim 1 or 8, is characterized in that, in described step S4, after described joint Connection Step, and before obtaining described amplicon library, also comprises and repairs to the band connecting joint " A " step that product carries out purifying.
10. an amplicon library, is characterized in that, adopts the construction process according to any one of claim 1 to 9 to build and forms.
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